Polarized exocytosis is fundamental to a wide range of physiological processes such as the release of neurotransmitters and the establishment of epithelial asymmetry. The budding yeast Saccharomyces cerevisiae grows asymmetrically, which requires polarized exocytosis for the incorporation of proteins and lipids to the daughter cell membrane for surface expansion and cell growth. This property, combined with its powerful genetics, makes the budding yeast an excellent model organism to study the molecular basis of polarized exocytosis. The exocyst is an evolutionarily conserved octameric protein complex that tethers post-Golgi secretory vesicles to specific domains of the plasma membrane for exocytosis. Our goal is to understand the molecular mechanism of polarized exocytosis mediated by the exocyst complex. Central to this question are how the exocyst is targeted to the plasma membrane, and how its function is spatially and kinetically regulated there. We hypothesize that the exocyst directly binds to the membrane through its interaction with phosphatidylinositol 4,5- bisphosphate (PI(4,5)P2), and the assembly of the exocyst complex mediates the tethering of post- Golgi secretory vesicles to the plasma membrane for exocytosis. In particular, the dual interactions of the exocyst component Sec3 with PI(4,5)P2 and Cdc42, a member of the Rho family of small GTPases, play important roles in exocyst targeting and function at the plasma membrane. Here we propose to analyze the molecular interactions of Cdc42 and phospholipids with Sec3 using biochemical methods. In addition we will determine the roles of Cdc42 and PI(4,5)P2 in the polarized localization of the exocyst, exocytosis, and cell morphogenesis. Finally, using a combination of biochemical, genetic, and cell biological approaches, we plan to characterize the novel EXR (Exocytosis Regulators) genes recently identified from our genetic screen for proteins that spatially and kinetically regulate the exocyst function in cells. Overall these studies will help us elucidate the molecular basis and regulatory mechanisms of exocytosis, and contribute to our understanding of diseases such as neurological degeneration, polycystic kidney diseases, and cancer. PUBLIC HEALTH REVELANCE: Polarized exocytosis is fundamental to many physiological processes such as neurotransmission, embryogenesis, and the establishment of epithelial cell asymmetry in kidney. Studying the molecular basis of polarized exocytosis will help us understand these physiological processes and shed light on the pathogenesis of diseases such as diabetes, neurological disorders, and cancer.